Impairments in skeletal muscle are a major contributor to the work intolerance and morbidity of patients with congestive heart failure (CHF). The fast-twitch skeletal muscle sarcoplasmic reticulum calcium ATPase (SERCA1) is a calcium transport pump whose expression is markedly decreased in CHF. The goal of this research is to elucidate the molecular mechanism and functional significance of decreased SERCA1 expression in skeletal muscle of rats with CHF. Aim 1 is to determine the contribution of decreased SERCA 1 transcription rate to decreases in SERCA 1 mRNA expression in skeletal muscle from rats with CHF. Transcription rate will be measured using a new, highly sensitive approach in which the SERCA precursor mRNA/mature mRNA ratio will be compared in tibialis anterior and diaphragm muscle from control and CHF rats using reverse transcriptase-polymerase chain reaction. Aim 2 is to study the mechanism by which SERCA 1 gene transcription is regulated by CHF. The cis-elements involved in SERCA 1 downregulation will be identified by deletion analysis. This will involve injecting the tibialis anterior muscle with plasmid DNA reporter constructs of the 5' flanking region of the SERCA 1 gene. Gel shift assay will detect and characterize the DNA binding protein(s) involved in transcriptional regulation and one-hybrid assays will identify novel DNA binding protein(s). If decreases in SERCA 1 mRNA expression are not predominately mediated by decreased transcription rate, then other pretranslational sites of control such as SERCA mRNA stability will be studied using in vitro assays of mRNA degradation and RNA-protein interactions. Aim 3 is to determine the functional significance of decreased SERCA expression in CHF. SERCA 1 expression will be "knocked down" in control muscles and overexpressed in CHF muscles by in vivo injection of appropriate plasmid DNA. SERCA1 protein expression will be quantified and localized by immunochemical assays and then related to parameters that reflect SERCA 1 function such as SERCA activity, SR calcium uptake rate and muscle relaxation time. Basic knowledge of the molecular regulation of SERCA gene expression in CHF can be applied towards more effective therapeutics in addition to furthering our understanding of the influence of SERCA 1 gene expression on skeletal muscle function.